Protein-substrate interactions are at the heart of biochemical events that govern signaling pathways and control physiological responses. These interactions dictate specificity in enzymatic reactions and are central to the control of biological responses, albeit, induced by endogenous compounds or drugs. A complete and thorough understanding of protein-substrate interactions is fundamental to the study of biological processes. As a model, we have undertaken a comprehensive study of wavelength regulation in rhodopsin. This is a remarkable example of protein-substrate interaction in which different proteins elicit different properties from the same substrate, namely 11-cis-retinal. The aim of our research is to understand the effect of different amino acid residues interacting with the retinal protonated Schiff base that ultimately lead to differing wavelength maxima of different pigments. We propose a novel strategy to study this unique phenomenon based on generating protonated Schiff base of 11-cis-retinal in protein environments that we will engineer to mimic rhodopsin, however, with the advantage of having at hand their 3-dimensional picture. This will allow us to rationally propose mutagenesis of amino acid residues in close contact with the bound substrate and probe the origins of wavelength regulation. We are also engaged in the synthesis of 13C-labeled retinals to probe the conformational aspects of wavelength regulation. Our second aim proposes to develop these rhodopsin protein mimics as colorimetric protein fusion tags, which are highly desired by the biotechnology field. We plan to demonstrate the utility of our rhodopsin protein mimics as ideal protein fusion tags with their small size, large binding cavity, tunable wavelength, and on/off activation; i.e., addition of chromophore will turn on the color. The small and soluble nature of the proposed proteins along with their unique 13-barrel cavity should allow binding of a variety of chromophores. The ability to vary the pigments spectral characteristics, either through various bound chromophores and/or tuning of the protein interactions with the bound chromophore, can lead to a variety of pigmented protein tags.